Abstract

The accumulation of amyloid-β (Aβ) peptides in the brain of patients with Alzheimer’s disease (AD) may arise from an imbalance between Aβ production and clearance. Overexpression of the Aβ-degrading enzyme neprilysin in brains of human amyloid precursor protein (hAPP) transgenic mice decreases overall Aβ levels and amyloid plaque burdens. Because AD-related synaptic and cognitive deficits appear to be more closely related to Aβ oligomers than to plaques, it is important to determine if increased neprilysin activity also diminishes the levels of pathogenic Aβ oligomers and related neuronal deficits in vivo. To address this question, we crossed hAPP transgenic mice with neprilysin transgenic mice and analyzed their offspring. Neprilysin overexpression reduced soluble Aβ levels by 50% and effectively prevented early Aβ deposition in the neocortex and hippocampus. However, it did not reduce levels of Aβ trimers and Aβ*56 or improve deficits in spatial learning and memory. The differential effect of neprilysin on plaques and oligomers suggests that neprilysin-dependent degradation of Aβ affects plaques more than oligomers and that these structures may form through distinct assembly mechanisms. Neprilysin’s inability to prevent learning and memory deficits in hAPP mice may be related to its inability to reduce pathogenic Aβ oligomers. Reduction of Aβ oligomers will likely be required for anti-Aβ treatments to improve cognitive functions.

Introduction

Alzheimer’s disease (AD) is the most prevalent and costly neurodegenerative disorder (Ferri et al., 2005). Although major efforts have been made to elucidate its pathogenesis, there still are no strategies to effectively treat or prevent AD. A key factor in AD pathogenesis is the amyloid-β (Aβ) peptide, which is generated through proteolytic cleavage of the amyloid precursor protein (APP) by β- and γ-secretases (Blennow et al., 2006; Roberson and Mucke, 2006). Rare forms of autosomal dominant familial AD (FAD) have been conceptually informative as they are linked to mutations in human APP (hAPP) or components of the γ-secretase complex (Presenilin 1 and Pensenilin 2) that increase the absolute or relative abundance of the 42–amino acid form of Aβ (Aβ1-42), which aggregates more readily and is more toxic than Aβ1-40 (Blennow et al., 2006; Roberson and Mucke, 2006). Transgenic mice expressing FAD-mutant hAPP produce high levels of Aβ1-42 and develop age-dependent cognitive deficits and neuropathological alterations similar to those found in AD patients (Kobayashi and Chen, 2005; Palop et al., 2006).

Materials and Methods

Animals

Mice used in this study included non-transgenic (NTG) and heterozygous transgenic (TG) mice from hAPP lines J9 and J20, which express hAPP with the Swedish (K670N, M671L) and Indiana (V717F) FAD mutations (hAPPFAD) directed by the platelet-derived growth factor β chain promoter (Rockenstein et al., 1995; Mucke et al., 2000; Palop et al., 2003). These lines have been crossed for >10 generations onto the C57BL/6J background using NTG mice from the Jackson Laboratory (Bar Harbor, ME). For some experiments, hAPP-J20 TG C57BL/6J mice were crossed with NTG DBA2J mice from the Jackson Laboratory to establish hAPP-J20 mice on the B6D2 F1 hybrid background. Mice overexpressing human NEP directed by the calcium/calmodulin-dependent kinase IIα promoter (Leissring et al., 2003) were on the C57BL/6J background. Unless indicated otherwise, offspring from the hAPP-J20 x NEP cross were on the same background. Some experiments were carried out in a mixed B6D2 background to allow for more direct comparisons with previous reports (Leissring et al., 2003; Poirier et al., 2006). After behavioral testing (see below), mice were deeply anesthetized followed by transcardial flush-perfusion with 0.9% saline and tissue isolation. All experiments were approved by the Institutional Animal Care and Use Committee of the University of California, San Francisco.

Open field

Spontaneous locomotor activity in an open field was measured in an automated Flex-Field/Open Field Photobeam Activity System (San Diego Instruments, San Diego, CA). Mice were placed in one of four identical clear plastic chambers (41 x 41 x 30 cm) for 15 min, with two 16 x 16 photobeam arrays detecting horizontal and vertical movements. Total movements (ambulations) in the outer periphery and center of the open field were reported. The apparatus was thoroughly cleaned with 70% ethanol between trials.

Elevated plus maze

The elevated plus maze consisted of two open (without walls) and two enclosed (with walls) arms elevated 63 cm above the ground (Hamilton-Kinder, Poway, CA). Mice were first allowed to habituate in the testing room under dim light for 30 min before testing. During testing, mice were placed at the junction between the open and closed arms of the plus maze and allowed to explore for 10 min. As a measure of general activity, total distance traveled and time spent in both the open and closed arms were calculated based on infrared photobeam breaks. In addition, the ratios of the distance traveled in the open arms over the total distance traveled in both open and closed arms were determined as a measure of the balance between exploratory drive and anxiety. The maze was thoroughly cleaned with 70% ethanol between testing sessions.

Morris water maze

The water maze consisted of a pool (122 cm diameter) filled with water (18 ± 1°C) made opaque with non-toxic white tempera paint powder and was placed in a room surrounded by distinct extramaze cues. Two identical pools were used in two different rooms, each with distinct extramaze cues. Pretraining and visible platform training were performed in one room, and hidden platform training and probe trials were performed in the other room. Mice were first given four pretraining trials in which they had to swim down a rectangular channel (15 cm x 122 cm) and mount a platform hidden 1.5 cm below the water surface at the end of the channel. The following day, mice were trained in the actual circular water maze. For cued training, the platform (14 cm x 14 cm) was submerged (1.5 cm) but visibly cued with a black and white striped mast (15 cm in height). On three consecutive days, mice received two training sessions per day separated by 3–4 hours. Each session consisted of two training trials with an intertrial interval of 10 min. For each session, the platform was moved to a new location, and the drop location was changed semi-randomly between trials. Mice were then moved to the second room for hidden platform training, in which a similar platform was submerged (1.5 cm) and not visibly cued. On five consecutive days, mice received two training sessions separated by 3–4 h. Each session consisted of three trials (10 min between trials). The platform location remained the same throughout the hidden platform training, but the drop location was changed semi-randomly between trials. Mice had 60 sec to locate the platform during training. Mice that did not find the platform were guided to it and placed on it for 10 sec. Spatial probe trial, during which the platform was removed and mice were allowed to swim in the pool for 60 sec, were carried out 16–18 h after the third day (beginning of Day 4) and fifth day (beginning of Day 6) of training. Escape latencies, swim paths, swim speeds, percent time spent in each quadrant, and platform crossings were recorded with an EthoVision video tracking system (Noldus, Wageningen, Netherlands).

ELISA and western blot analyses

The left hemibrain was snap-frozen on dry ice and stored at −80°C. Whole hemibrains were either used as such or dissected into two preparations: 1) hippocampal formation and 2) forebrain without hippocampal formation, thalamus, cerebellum and brainstem (“cortex-plus”). For the Aβ ELISA measurements of human Aβ1-x and Aβ1-42, hippocampal or cortex-plus tissues were homogenized in 5 M guanidine buffer as described (Mucke et al., 2000).

Immunohistochemistry

The right hemibrain was drop-fixed in 4% paraformaldehyde for 48 h at 4°C, rinsed in phosphate buffered saline (PBS), and placed in a 30% sucrose solution for at least 24 h at 4°C. Immunostaining was performed on sliding microtome sections (30 μm) by the standard avidin-biotin/peroxidase method as described (Palop et al., 2003). Briefly, after quenching endogenous peroxidase activity and blocking non-specific binding, floating sections were incubated with anti-calbindin (1:30,000; Swant, Bellinzona, Switzerland), anti-Fos (Ab-5, 1:10,000, Calbiochem), or anti-Aβ (3D6, 1:400 dilution of 1.97 mg/ml biotinylated stock, Elan Pharmaceuticals) antibodies at 4°C. For the first two antibodies, sections were subsequently incubated with biotinylated goat anti-rabbit (1:200, Vector Laboratories, Burlingame, CA) secondary antibodies for 1 h at room temperature. Diaminobenzidine was used as a chromagen. Images were acquired on a BX-60 microscope (Olympus, Melville, NY) and recorded with a DEI-470 digital camera (Optronics, Goleta, CA). Immunoreactivities for calbindin, Fos, and Aβ were quantitated as described (Mucke et al., 2000; Palop et al., 2003; Chin et al., 2005a).

Statistical analysis

Statistical analyses were performed with Statview 5.0 (SAS Institute, Cary, NC). Differences between means were assessed, as appropriate, by Student’s t-test, one- or two-way ANOVA with or without repeated measures, followed by Fisher’s PLSD or Tukey’s post-hoc tests. Correlations were assessed by simple linear regression analysis. Kaplan-Meier statistical methods were used to determine survival rates and hazard plots, followed by Logrank (Mantel-Cox) post hoc tests.

NEP overexpression does not prevent learning and memory deficits in hAPP mice

To determine if the NEP-mediated reductions in overall Aβ levels and in Aβ deposits in hAPP-J20/NEP mice were sufficient to prevent learning and memory deficits, we tested 3–5-month-old NTG, NEP, hAPP-J20, and hAPP-J20/NEP mice in a Morris water maze. On both the C57BL/6J and the B6D2 backgrounds, hAPP-J20 and hAPP-J20/NEP mice had similar difficulties with learning to locate the hidden platform in the spatial component of the task, as compared with NTG controls (Fig. 2A, B). On the C57BL/6J, but not the B6D2, background, hAPP-J20 and hAPP-J20/NEP mice also showed some deficits in the cued component of the task, although by the last day of cued training all mice performed similarly well (Fig. 2A, B). NEP mice performed as well as NTG mice in both components of the test. Comparison of swim speeds during the hidden platform training revealed no significant differences between the groups (data not shown).

To assess spatial memory formation and retention, we removed the platform for a probe trial at the beginning of the fourth day of hidden platform training. Regardless of strain background, hAPP-J20 and hAPP-J20/NEP mice spent less time searching in the target quadrant (Fig. 2C, D) and crossed over the target location less frequently (Fig. 2E, F) than NTG and NEP mice. hAPP-J20 and hAPP-J20/NEP mice did not differ significantly with respect to either outcome measure. To assess whether the different groups of mice would benefit differentially from prolonged training, we continued the hidden platform training for another 2 days and administered another probe trial 16–18 h after the last hidden platform training trial. The continued training increased target quadrant preference for both hAPP-J20 and hAPP-J20/NEP mice (Fig. 2G, H). On the C57BL/6J background, both groups still showed comparable impairments in target crossings (Fig. 2I). On the B6D2 background, only hAPP-J20 mice were impaired in this measure (Fig. 2J), although the difference between hAPP-J20 and hAPP-J20/NEP mice was not statistically significant.

Overexpression of NEP does not reduce levels of Aβ*56 or Aβ trimers. Different Aβ assemblies were measured in 5–6-month-old hAPPFAD TG C57BL/6J mice from line J20 and from the lower expresser line J9. A, Representative...

Because smaller Aβ assemblies, such as dimers and trimers, can also cause functional neuronal impairments and memory deficits (Townsend et al., 2006; Shankar et al., 2007; Klyubin et al., 2008; Shankar et al., 2008), we wanted to determine if NEP overexpression reduced the levels of these species. Aβ trimer levels were detected in the same samples used in Fig. 4A. Tissue lysates were immunoprecipitated with the anti-Aβ antibody 4G8 and then examined by western blotting with the anti-Aβ antibodies 3D6 (Fig. 4B, D) or 6E10 (data not shown). Aβ trimer levels were comparable in hAPP-J20 and hAPP-J20/NEP mice (Fig. 4B, D and data not shown).

Aβ*56 and Aβ trimers were already detectable in hAPP-J20 mice at 1.5 months of age (data not shown), suggesting that both might contribute to the subsequent development of behavioral deficits. In contrast, Aβ dimers were undetectable in whole hemibrains of 3–8-month-old hAPP-J20 mice (Fig. 4E), making it unlikely that these assemblies contribute to the prominent cognitive deficits seen in these mice. Aβ dimers were detectable only in old hAPP-J20 mice (Fig. 4E), which have extensive plaque burdens (Roberson et al., 2007).

Because NEP overexpression reduced total Aβ levels in hAPP mice from the high-expresser line J20 (Fig. 1D) to levels found in our lower expressing hAPP line J9 (Mucke et al., 2000), we analyzed levels of Aβ*56 and Aβ trimers in the latter line. Levels of Aβ*56 (Fig. 4A, C) and Aβ trimers (Fig. 4B, D) were significantly lower in hAPP-J9 mice than in hAPP-J20 and hAPP-J20/NEP mice. Thus, overall Aβ levels are likely a partial, but not the sole, determinant of Aβ*56 and Aβ trimer levels.

NEP overexpression prevents hyperactivity but not alterations in anxiety/exploration-related behavior in hAPP mice

Overexpression of NEP prevents hyperactivity but does not alter elevated plus maze abnormalities in hAPP-J20 mice. Total activity in the periphery of an open field (A, B) and the proportional distance traveled in the open arms of an elevated plus maze...

Effect of NEP overexpression on premature mortality in hAPP mice

NEP overexpression has been reported to diminish premature mortality during the first 6 months of life in hAPP-J20 mice on the B6D2 background (Leissring et al., 2003). To confirm and extend these results, we compared the survival of NTG, hAPP-J20, NEP, and hAPP-J20/NEP mice on the B6D2 and the C57BL/6J backgrounds between postnatal day 14 and 17 months. Surprisingly, overexpression of NEP in hAPP-J20/NEP mice did not improve premature mortality on the B2D6 background and actually worsened it on the C57BL/6 background (Fig. 6), as compared with hAPP-J20 mice. Premature mortality of hAPP-J20/NEP mice was greater in the C57BL/6J background than in the B2D6 background, whereas premature mortality of hAPP-J20 mice was not affected by background (Fig. 6). In the absence of hAPP/Aβ, overexpression of NEP did not affect survival in either background (Fig. 6).

To determine if the increased mortality of hAPP-J20/NEP mice on the C57BL/6J background was caused by an interaction between NEP and high levels of either hAPP or Aβ, we crossed NEP mice with TG mice expressing wildtype hAPP at high levels (line I5) or with TG mice expressing FAD-mutant hAPP at lower levels (line J9) (Mucke et al., 2000; Palop et al., 2003). Average levels of hAPP expression are roughly comparable in TG mice from lines I5 and J20, whereas Aβ levels are much lower in I5 than J20 mice (Mucke et al., 2000; Meilandt et al., 2008). Average levels of hAPP and Aβ in J9 mice are roughly half of those in J20 mice (Mucke et al., 2000). Overexpression of NEP had no effect on survival in hAPP-I5 mice and hAPP-J9 mice (Supplemental Fig. 2).

NEP’s inability to reduce Aβ trimers or cognitive deficits in vivo agrees with its inability to degrade such Aβ assemblies in vitro (Leissring et al., 2003) and with deleterious effects of Aβ trimers on neuronal function (Townsend et al., 2006). The putative dodecamer Aβ*56 that correlates with memory deficits in hAPP mice (Lesné et al., 2006; Cheng et al., 2007) is also likely resistant to NEP as its levels were similar in hAPP-J20 and hAPP-J20/NEP mice. Because Aβ dimers appeared only months after functional deficits, they are unlikely to contribute to behavioral alterations in hAPP-J20 mice. In contrast, Aβ*56 and Aβ trimers occurred before these alterations emerged and, thus, are a likelier cause of neuronal dysfunction in this model.

In contrast to our study, others reported that NEP overexpression prevents learning and memory deficits in hAPP mice (Poirier et al., 2006). Different levels, times, and patterns of transgene expression may account for the discrepancy. Brain NEP activity was ~30-fold higher than controls in their NEP TG line (Poirier et al., 2006) and ~8-fold higher in the line we used (Leissring et al., 2003). The CamKII promoter used in our line directs expression late in postnatal development (P3–P15) and predominantly within forebrain excitatory neurons (Mayford et al., 1997; Solà et al., 1999). The PrP promoter used in their line directs expression at E13.5 and in additional neuronal populations, including inhibitory interneurons (Manson et al., 1992; Weber et al., 2001; Bailly et al., 2004). Because Poirier and colleagues did not measure Aβ oligomer levels, it is unclear if the behavioral benefits they reported were associated with a reduction in these moieties.

NEP overexpression prevented hyperactivity in hAPP-J20 mice. Because Aβ oligomer levels in hAPP-J20 and hAPP-J20/NEP mice were comparable, hyperactivity is more likely caused by monomeric Aβ than Aβ*56 or Aβ trimers. It also does not correlate with Aβ*56 levels in hAPP mice (Cheng et al., 2007). Although another Aβ assembly might produce hyperactivity, we doubt that plaques do, as hyperactivity occurs before plaques form.

Several hAPP lines, including hAPP-J20, show decreased open arm avoidance in the elevated plus maze (Chin et al., 2005b; Kobayashi and Chen, 2005; Cheng et al., 2007; Roberson et al., 2007). NEP overexpression did not prevent this abnormality in hAPP-J20 mice, suggesting it may be caused by Aβ*56 or Aβ trimers rather than monomers or plaques. However, decreased open arm avoidance did not correlate with forebrain Aβ*56 levels (Cheng et al., 2007). Because quantifying Aβ*56 levels in small samples is difficult, we cannot exclude effects of Aβ*56 or Aβ trimers on specific brain regions, such as the amygdala; other Aβ assemblies and hAPP metabolites may contribute also.

Comparing hAPP-J20/NEP mice with hAPP-J20 and hAPP-J9 mice may yield insights into formation and accumulation of pathogenic Aβ oligomers in the brain. Overall Aβ levels in hAPP-J20 mice were twice those in hAPP-J9 mice, and Aβ*56 levels were significantly higher in hAPP-J20 than hAPP-J9 mice, suggesting that overall Aβ levels do affect accumulation of pathogenic oligomers. Overexpression of NEP in young hAPP-J20/NEP mice decreased Aβ monomers and overall Aβ levels by >50%, to levels below those found in hAPP-J9 mice. This Aβ reduction decreased Aβ deposition, but had no effect on oligomers. Indeed, Aβ*56 and Aβ trimer levels were comparable in hAPP-J20 and hAPP-J20/NEP mice and lower in hAPP-J9 mice. Thus, formation of Aβ oligomers and formation/deposition of Aβ fibrils may follow distinct pathways in vivo.

Why does NEP overexpression reduce formation of plaques but not Aβ*56 and trimers? Since Aβ dimer accumulation closely parallels the appearance of SDS-insoluble fibrillar Aβ (Kawarabayashi et al., 2004), formation of dimers and plaques may be causally related. Dimers may represent a critical nucleation step towards fibrillization (Congdon et al., 2008), whereas trimers may be critical for the formation of Aβ*56 and other higher-order nonfibrillar assemblies. Assuming NEP reduces Aβ dimers more than trimers, this differential assembly hypothesis could explain why NEP overexpression reduces plaques but not Aβ*56. If Aβ oligomer formation required fewer Aβ monomers than plaque formation, overall Aβ levels in hAPP-J20/NEP mice may not be low enough to affect oligomer formation. We consider this unlikely, though, because overall Aβ levels were slightly lower in hAPP-J20/NEP than hAPP-J9 mice, whereas Aβ oligomer levels were higher in hAPP-J20/NEP mice.

Interestingly, NEP ablation in line APP23 worsened behavioral abnormalities and was associated with subtle increases in trimers and tetramers (Huang et al., 2006). Therefore, NEP may have a small effect on Aβ trimers and higher-order oligomers. NEP ablation may increase Aβ oligomer levels above a pathogenic threshold in APP23 mice, bringing out functional deficits. Aβ oligomer levels in hAPP-J20 mice may be so high that NEP overexpression cannot reduce them below this threshold.

Our study suggests increasing NEP activity in the context of high Aβ oligomer levels may have limited therapeutic benefit and could be associated with untoward side effects. Identifying and harnessing alternative Aβ-degrading enzymes that more effectively destroy pathogenic oligomers are more promising objectives.

Acknowledgments

We thank M. Leissring and D. Selkoe for neprilysin transgenic mice; D. Walsh and G. Shankar for advice on dimer measurements; P. Seubert for anti-Aβ antibodies; E. Koo for the CT-15 antibody; J. Palop for helpful comments on the manuscript; H. Solanoy, X. Wang and D. Kim for technical support; G. Howard and S. Ordway for editorial review; J. Carroll for preparation of graphics; and D. McPherson and T. Hamto for administrative assistance. The study was supported by NIH Grants AG011385 and NS041787 to L.M. and RR018928 to the J. David Gladstone Institutes.

Kanemitsu H, Tomiyama T, Mori H. Human neprilysin is capable of degrading amyloid β peptide not only in the monomeric form but also the pathological oligomeric form. Neurosci Lett. 2003;350:113–116.[PubMed]